Cristian Rodriguez-Aguayo

Integrated Analyses Identify a Master MicroRNA Regulatory Network for the Mesenchymal Subtype in Serous Ovarian Cancer

Integrated genomic analyses revealed a miRNA-regulatory network that further defined a robust integrated mesenchymal subtype associated with poor overall survival in 459 cases of serous ovarian cancer (OvCa) from The Cancer Genome Atlas and 560 cases from independent cohorts. Eight key miRNAs, including miR-506, miR-141, and miR-200a, were predicted to regulate 89% of the targets in this network. Follow-up functional experiments illustrate that miR-506 augmented E-cadherin expression, inhibited cell migration and invasion, and prevented TGFβ-induced epithelial-mesenchymal transition by targeting SNAI2, a transcriptional repressor of E-cadherin. In human OvCa, miR-506 expression was correlated with decreased SNAI2 and VIM, elevated E-cadherin, and beneficial prognosis. Nanoparticle delivery of miR-506 in orthotopic OvCa mouse models led to E-cadherin induction and reduced tumor growth.

Tumour angiogenesis regulation by the miR-200 family

The miR-200 family is well known to inhibit the epithelial-mesenchymal transition, suggesting it may therapeutically inhibit metastatic biology. However, conflicting reports regarding the role of miR-200 in suppressing or promoting metastasis in different cancer types have left unanswered questions. Here we demonstrate a difference in clinical outcome based on miR-200s role in blocking tumour angiogenesis. We demonstrate that miR-200 inhibits angiogenesis through direct and indirect mechanisms by targeting interleukin-8 and CXCL1 secreted by the tumour endothelial and cancer cells. Using several experimental models, we demonstrate the therapeutic potential of miR-200 delivery in ovarian, lung, renal and basal-like breast cancers by inhibiting angiogenesis. Delivery of miR-200 members into the tumour endothelium resulted in marked reductions in metastasis and angiogenesis, and induced vascular normalization. The role of miR-200 in blocking cancer angiogenesis in a cancer-dependent context defines its utility as a potential therapeutic agent.

Metabolic shifts toward glutamine regulate tumor growth, invasion and bioenergetics in ovarian cancer

Glutamine can play a critical role in cellular growth in multiple cancers. Glutamine‐addicted cancer cells are dependent on glutamine for viability, and their metabolism is reprogrammed for glutamine utilization through the tricarboxylic acid (TCA) cycle. Here, we have uncovered a missing link between cancer invasiveness and glutamine dependence. Using isotope tracer and bioenergetic analysis, we found that low‐invasive ovarian cancer (OVCA) cells are glutamine independent, whereas high‐invasive OVCA cells are markedly glutamine dependent. Consistent with our findings, OVCA patients’ microarray data suggest that glutaminolysis correlates with poor survival. Notably, the ratio of gene expression associated with glutamine anabolism versus catabolism has emerged as a novel biomarker for patient prognosis. Significantly, we found that glutamine regulates the activation of STAT3, a mediator of signaling pathways which regulates cancer hallmarks in invasive OVCA cells. Our findings suggest that a combined approach of targeting high‐invasive OVCA cells by blocking glutamines entry into the TCA cycle, along with targeting low‐invasive OVCA cells by inhibiting glutamine synthesis and STAT3 may lead to potential therapeutic approaches for treating OVCAs.

Src activation by adrenoreceptors is a key switch for tumour metastasis

Norepinephrine (NE) can modulate multiple cellular functions important for cancer progression; however, how this single extracellular signal regulates such a broad array of cellular processes is unknown. Here, we identify Src as a key regulator of phosphoproteomic signaling networks activated in response to beta-adrenergic signaling in cancer cells. These results also identify a new mechanism of Src phosphorylation that mediates beta-adrenergic/PKA regulation of downstream networks, thereby enhancing tumor cell migration, invasion and growth. In human ovarian cancer samples, high tumoral NE levels were correlated with high pSrcY419 levels. Moreover, among cancer patients, the use of beta blockers was significantly associated with reduced cancer-related mortality. Collectively, these data provide a pivotal molecular target for disrupting neural signaling in the tumor microenvironment.

Hematogenous Metastasis of Ovarian Cancer: Rethinking Mode of Spread

Ovarian cancer has a clear predilection for metastasis to the omentum, but the underlying mechanisms involved in ovarian cancer spread are not well understood. Here, we used a parabiosis model that demonstrates preferential hematogenous metastasis of ovarian cancer to the omentum. Our studies revealed that the ErbB3-neuregulin 1 (NRG1) axis is a dominant pathway responsible for hematogenous omental metastasis. Elevated levels of ErbB3 in ovarian cancer cells and NRG1 in the omentum allowed for tumor cell localization and growth in the omentum. Depletion of ErbB3 in ovarian cancer impaired omental metastasis. Our results highlight hematogenous metastasis as an important mode of ovarian cancer metastasis. These findings have implications for designing alternative strategies aimed at preventing and treating ovarian cancer metastasis.

Hypoxia-mediated downregulation of miRNA biogenesis promotes tumour progression

Cancer-related deregulation of miRNA biogenesis has been suggested, but the underlying mechanisms remain elusive. Here we report a previously unrecognized effect of hypoxia in the downregulation of Drosha and Dicer in cancer cells that leads to dysregulation of miRNA biogenesis and increased tumour progression. We show that hypoxia-mediated downregulation of Drosha is dependent on ETS1/ELK1 transcription factors. Moreover, mature miRNA array and deep sequencing studies reveal altered miRNA maturation in cells under hypoxic conditions. At a functional level, this phenomenon results in increased cancer progression in vitro and in vivo, and data from patient samples are suggestive of miRNA biogenesis downregulation in hypoxic tumours. Rescue of Drosha by siRNAs targeting ETS1/ELK1 in vivo results in significant tumour regression. These findings provide a new link in the mechanistic understanding of global miRNA downregulation in the tumour microenvironment. MicroRNAs play important roles in the maintenance of cellular homeostasis through the post-transcriptional regulation of gene expression. Here, the authors implicate loss of the miRNA biogenesis factor Drosha and altered miRNA maturation in tumour progression under hypoxic conditions.

Enhancing chemotherapy response with sustained EphA2 silencing using multistage vector delivery

Purpose: RNA interference has the potential to specifically knockdown the expression of target genes and thereby transform cancer therapy. However, lack of effective delivery of siRNA has dramatically limited its in vivo applications. We have developed a multistage vector (MSV) system, composed of discoidal porous silicon particles loaded with nanotherapeutics, that directs effective delivery and sustained release of siRNA in tumor tissues. In this study, we evaluated therapeutic efficacy of MSV-loaded EphA2 siRNA (MSV/EphA2) with murine orthotopic models of metastatic ovarian cancers as a first step toward development of a new class of nanotherapeutics for the treatment of ovarian cancer. Experimental Design: Tumor accumulation of MSV/EphA2 and sustained release of siRNA from MSV were analyzed after intravenous administration of MSV/siRNA. Nude mice with metastatic SKOV3ip2 tumors were treated with MSV/EphA2 and paclitaxel, and therapeutic efficacy was assessed. Mice with chemotherapy-resistant HeyA8 ovarian tumors were treated with a combination of MSV/EphA2 and docetaxel, and enhanced therapeutic efficacy was evaluated. Results: Treatment of SKOV3ip2 tumor mice with MSV/EphA2 biweekly for 6 weeks resulted in dose-dependent (5, 10, and 15 μg/mice) reduction of tumor weight (36%, 64%, and 83%) and number of tumor nodules compared with the control groups. In addition, tumor growth was completely inhibited when mice were treated with MSV/EphA2 in combination with paclitaxel. Furthermore, combination treatment with MSV/EphA2 and docetaxel inhibited growth of HeyA8-MDR tumors, which were otherwise resistant to docetaxel treatment. Conclusion: These findings indicate that MSV/EphA2 merits further development as a novel therapeutic agent for ovarian cancer. Clin Cancer Res; 19(7); 1806–15. ©2013 AACR.

TP53 loss creates therapeutic vulnerability in colorectal cancer

TP53, a well-known tumour suppressor gene that encodes p53, is frequently inactivated by mutation or deletion in most human tumours. A tremendous effort has been made to restore p53 activity in cancer therapies. However, no effective p53-based therapy has been successfully translated into clinical cancer treatment owing to the complexity of p53 signalling. Here we demonstrate that genomic deletion of TP53 frequently encompasses essential neighbouring genes, rendering cancer cells with hemizygous TP53 deletion vulnerable to further suppression of such genes. POLR2A is identified as such a gene that is almost always co-deleted with TP53 in human cancers. It encodes the largest and catalytic subunit of the RNA polymerase II complex, which is specifically inhibited by α-amanitin. Our analysis of The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases reveals that POLR2A expression levels are tightly correlated with its gene copy numbers in human colorectal cancer. Suppression of POLR2A with α-amanitin or small interfering RNAs selectively inhibits the proliferation, survival and tumorigenic potential of colorectal cancer cells with hemizygous TP53 loss in a p53-independent manner. Previous clinical applications of α-amanitin have been limited owing to its liver toxicity. However, we found that α-amanitin-based antibody–drug conjugates are highly effective therapeutic agents with reduced toxicity. Here we show that low doses of α-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody lead to complete tumour regression in mouse models of human colorectal cancer with hemizygous deletion of POLR2A. We anticipate that inhibiting POLR2A will be a new therapeutic approach for human cancers containing such common genomic alterations.

Cystathionine Beta-Synthase (CBS) Contributes to Advanced Ovarian Cancer Progression and Drug Resistance

Background Epithelial ovarian cancer is the leading cause of gynecologic cancer deaths. Most patients respond initially to platinum-based chemotherapy after surgical debulking, however relapse is very common and ultimately platinum resistance emerges. Understanding the mechanism of tumor growth, metastasis and drug resistant relapse will profoundly impact the therapeutic management of ovarian cancer. Methods/Principal Findings Using patient tissue microarray (TMA), in vitro and in vivo studies we report a role of of cystathionine-beta-synthase (CBS), a sulfur metabolism enzyme in ovarian carcinoma. We report here that the expression of cystathionine-beta-synthase (CBS), a sulfur metabolism enzyme, is common in primary serous ovarian carcinoma. The in vitro effects of CBS silencing can be reversed by exogenous supplementation with the GSH and H2S producing chemical Na2S. Silencing CBS in a cisplatin resistant orthotopic model in vivo by nanoliposomal delivery of CBS siRNA inhibits tumor growth, reduces nodule formation and sensitizes ovarian cancer cells to cisplatin. The effects were further corroborated by immunohistochemistry that demonstrates a reduction of H&E, Ki-67 and CD31 positive cells in si-RNA treated as compared to scrambled-RNA treated animals. Furthermore, CBS also regulates bioenergetics of ovarian cancer cells by regulating mitochondrial ROS production, oxygen consumption and ATP generation. This study reports an important role of CBS in promoting ovarian tumor growth and maintaining drug resistant phenotype by controlling cellular redox behavior and regulating mitochondrial bioenergetics. Conclusion The present investigation highlights CBS as a potential therapeutic target in relapsed and platinum resistant ovarian cancer.

miR-205 acts as a tumour radiosensitizer by targeting ZEB1 and Ubc13

Tumor cells associated with therapy resistance (radioresistance and drug resistance) are likely to give rise to local recurrence and distant metastatic relapse. Recent studies revealed microRNA (miRNA)-mediated regulation of metastasis and epithelial-mesenchymal transition; however, whether specific miRNAs regulate tumor radioresistance and can be exploited as radiosensitizing agents remains unclear. Here we find that miR-205 promotes radiosensitivity and is downregulated in radioresistant subpopulations of breast cancer cells, and that loss of miR-205 is highly associated with poor distant relapse-free survival in breast cancer patients. Notably, therapeutic delivery of miR-205 mimics via nanoliposomes can sensitize the tumor to radiation in a xenograft model. Mechanistically, radiation suppresses miR-205 expression through ataxia telangiectasia mutated (ATM) and zinc finger E-box binding homeobox 1 (ZEB1). Moreover, miR-205 inhibits DNA damage repair by targeting ZEB1 and the ubiquitin-conjugating enzyme Ubc13. These findings identify miR-205 as a radiosensitizing miRNA and reveal a new therapeutic strategy for radioresistant tumors.